High resolution atomic force microscopy of double-stranded RNA.

Double-stranded (ds) RNA mediates the suppression of specific gene expression, it is the genetic material of a number of viruses, and a key activator of the innate immune response against viral infections. The ever increasing list of roles played by dsRNA in the cell and its potential biotechnological applications over the last decade has raised an interest for the characterization of its mechanical properties and structure, and that includes approaches using Atomic Force Microscopy (AFM) and other single-molecule techniques. Recent reports have resolved the structure of dsDNA with AFM at unprecedented resolution. However, an equivalent study with dsRNA is still lacking. Here, we have visualized the double helix of dsRNA under near-physiological conditions and at sufficient resolution to resolve the A-form sub-helical pitch periodicity. We have employed different high-sensitive force-detection methods and obtained images with similar spatial resolution. Therefore, we show here that the limiting factors for high-resolution AFM imaging of soft materials in liquid medium are, rather than the imaging mode, the force between the tip and the sample and the sharpness of the tip apex.

Amyloidogenesis of bacterial prionoid RepA-WH1 recapitulates dimer to monomer transitions of RepA in DNA replication initiation.

Most available structures of amyloids correspond to peptide fragments that self-assemble in extended cross ß sheets. However, structures in which a whole protein domain acts as building block of an amyloid fiber are scarce, in spite of their relevance to understand amyloidogenesis. Here, we use electron microscopy (EM) and atomic force microscopy (AFM) to analyze the structure of amyloid filaments assembled by RepA-WH1, a winged-helix domain from a DNA replication initiator in bacterial plasmids. RepA-WH1 functions as a cytotoxic bacterial prionoid that recapitulates features of mammalian amyloid proteinopathies. RepA are dimers that monomerize at the origin to initiate replication, and we find that RepA-WH1 reproduces this transition to form amyloids. RepA-WH1 assembles double helical filaments by lateral association of a single-stranded precursor built by monomers. Double filaments then associate in mature fibers. The intracellular and cytotoxic RepA-WH1 aggregates might reproduce the hierarchical assembly of human amyloidogenic proteins.

Sequence-specific interactions of Rep proteins with ssDNA in the AT-rich region of the plasmid replication origin.

The DNA unwinding element (DUE) is a sequence rich in adenine and thymine residues present within the origin region of both prokaryotic and eukaryotic replicons. Recently, it has been shown that this is the site where bacterial DnaA proteins, the chromosomal replication initiators, form a specific nucleoprotein filament. DnaA proteins contain a DNA binding domain (DBD) and belong to the family of origin binding proteins (OBPs). To date there has been no data on whether OBPs structurally different from DnaA can form nucleoprotein complexes within the DUE. In this work we demonstrate that plasmid Rep proteins, composed of two Winged Helix domains, distinct from the DBD, specifically bind to one of the strands of ssDNA within the DUE. We observed nucleoprotein complexes formed by these Rep proteins, involving both dsDNA containing the Rep-binding sites (iterons) and the strand-specific ssDNA of the DUE. Formation of these complexes required the presence of all repeated sequence elements located within the DUE. Any changes in these repeated sequences resulted in the disturbance in Rep-ssDNA DUE complex formation and the lack of origin replication activity in vivo or in vitro.

DNA origami nanopores for controlling DNA translocation.

We combine DNA origami structures with glass nanocapillaries to reversibly form hybrid DNA origami nanopores. Trapping of the DNA origami onto the nanocapillary is proven by imaging fluorescently labeled DNA origami structures and simultaneous ionic current measurements of the trapping events. We then show two applications highlighting the versatility of these DNA origami nanopores. First, by tuning the pore size we can control the folding of dsDNA molecules ("physical control"). Second, we show that the specific introduction of binding sites in the DNA origami nanopore allows selective detection of ssDNA as a function of the DNA sequence ("chemical control").

Electrostatic binding and hydrophobic collapse of peptide-nucleic acid aggregates quantified using force spectroscopy.

Knowledge of the mechanisms of interaction between self-aggregating peptides and nucleic acids or other polyanions is key to the understanding of many aggregation processes underlying several human diseases (e.g., Alzheimer's and Parkinson's diseases). Determining the affinity and kinetic steps of such interactions is challenging due to the competition between hydrophobic self-aggregating forces and electrostatic binding forces. Kahalalide F (KF) is an anticancer hydrophobic peptide that contains a single positive charge that confers strong aggregative properties with polyanions. This makes KF an ideal model to elucidate the mechanisms by which self-aggregation competes with binding to a strongly charged polyelectrolyte such as DNA. We use optical tweezers to apply mechanical forces to single DNA molecules and show that KF and DNA interact in a two-step kinetic process promoted by the electrostatic binding of DNA to the aggregate surface followed by the stabilization of the complex due to hydrophobic interactions. From the measured pulling curves we determine the spectrum of binding affinities, kinetic barriers, and lengths of DNA segments sequestered within the KF-DNA complex. We find there is a capture distance beyond which the complex collapses into compact aggregates stabilized by strong hydrophobic forces and discuss how the bending rigidity of the nucleic acid affects this process. We hypothesize that within an in vivo context, the enhanced electrostatic interaction of KF due to its aggregation might mediate the binding to other polyanions. The proposed methodology should be useful to quantitatively characterize other compounds or proteins in which the formation of aggregates is relevant.

AFM volumetric methods for the characterization of proteins and nucleic acids.

The atomic force microscope overestimates lateral dimensions and underestimates heights of nanometer size objects such as proteins and nucleic acids. This has made researchers cautious of AFM measurements, even though there is no other technique capable of measuring topography with sub-nanometer precision. Nevertheless, several approaches for determining the stoichiometry of protein and protein-DNA complexes have been developed which show that, although the absolute values may be incorrect, the AFM volume is essentially proportional to the mass. This has allowed the determination of the mass of protein complexes with the help of a calibration curve. Here we review the main techniques for AFM volume measurements and detail a methodology that significantly reduces the associated errors. This method uses a fragment of DNA as a fiducial marker by which the volume of a protein is normalized. The use of fiducial markers co-adsorbed together with the protein of interest minimizes the contribution of tip-induced artifacts as they affect both the object of interest and the marker. Finally, we apply this method to the measurement of the length of single-stranded DNA. A linear relationship between length and volume was obtained, opening the door to studies of ssDNA intermediates formed during complex DNA transactions such as replication, recombination and repair.

Mechanical identities of RNA and DNA double helices unveiled at the single-molecule level.

Double-stranded (ds) RNA is the genetic material of a variety of viruses and has been recently recognized as a relevant molecule in cells for its regulatory role. Despite that the elastic response of dsDNA has been thoroughly characterized in recent years in single-molecule stretching experiments, an equivalent study with dsRNA is still lacking. Here, we have engineered long dsRNA molecules for their individual characterization contrasting information with dsDNA molecules of the same sequence. It is known that dsRNA is an A-form molecule unlike dsDNA, which exhibits B-form in physiological conditions. These structural types are distinguished at the single-molecule level with atomic force microscopy (AFM) and are the basis to understand their different elastic response. Force-extension curves of dsRNA with optical and magnetic tweezers manifest two main regimes of elasticity, an entropic regime whose end is marked by the A-form contour-length and an intrinsic regime that ends in a low-cooperative overstretching transition in which the molecule extends to 1.7 times its A-form contour-length. DsRNA does not switch between the A and B conformations in the presence of force. Finally, dsRNA presents both a lower stretch modulus and overstretching transition force than dsDNA, whereas the electrostatic and intrinsic contributions to the persistence length are larger.